1. Field of the Invention
This invention relates to devices and processes for hybridizing nucleic acid samples, and more particularly, to an automated device for hybridizing DNA microarrays.
2. Discussion
Use of DNA (deoxyribonucleic acid) microarrays provides a powerful technique to analyze expression of thousands of genes simultaneously. The technique includes immobilizing DNA samples from large numbers of genes on a solid substrate, such as a glass microscope slide. The DNA samples appear as an array of spots on the substrate, and one can determine the origin of a particular DNA sample by knowing its position in the array. The technique typically provides contacting the DNA microarray with RNA (ribonucleic acid) probes to detect specific nucleotide sequences in the DNA samples. To distinguish between different RNA probes, each is labeled with a tag that fluoresces at a wavelength that is unique for the particular probe.
Under proper conditions, the RNA probes will hybridize or bind to the immobilized DNA samples, resulting in hybrid DNA-RNA strands. For each of the immobilized DNA samples, and for a particular RNA probe, one can discern differences in hybridization among DNA samples by measuring the intensity and the wavelength dependence of fluorescence of each microarray element. In this way, one can determine whether gene expression levels vary among DNA samples. Thus, using DNA microarrays, one can learn much about expression of a large number of genes, and about comprehensive patterns of gene expression, using relatively small amounts of biological material.
Although DNA microarrays are powerful tools, instruments currently available to hybridize DNA microarrays need improvement. Most instruments that can process DNA microarrays have rudimentary temperature control. But nucleic acid hybridization demands precise temperature control. Rates of hybridization and equilibrium concentrations of hybrid DNA-RNA strands depend strongly on temperature and therefore accurate comparisons among hybridization experiments require that the experiments be run at the same temperature. In addition, precise temperature programming during an experiment is often critical to minimizing spurious probe-sample binding. For example, rapidly decreasing temperature following hybridizationxe2x80x94a process called step-wise probe annealingxe2x80x94reduces background binding.
Generally, instruments that can process DNA microarrays also lack an adequate system for controlling fluid contacting. During hybridization, the DNA microarray is immersed in a fluid that contains the RNA probes. The rate at which the probes bind to the DNA samples will depend, in part, on the concentration of the probes. However, the concentration of the probes near the immobilized DNA samples may be much different than the bulk concentration of the probes. Although agitating the fluid helps minimize concentration gradients between the bulk fluid and fluid next to the substrate surface, excessive fluid mixing may create high shearing and normal forces that may dislodge the DNA samples.
The present invention overcomes, or at least reduces, one or more of the problems set forth above.
The present invention provides a DNA hybridization apparatus capable of precise thermal and fluid control. The present invention is particularly useful when used in conjunction with DNA spotted glass slides (DNA microarrays). The apparatus can also be used for hybridizing other materials on other substrates. Multiple slides can be processed at one time (in parallel) or in rapid serial fashion. A fluid manifold allows for control of multiple fluids across the surface of each slide. All slides can contact the same sequence of fluids or may undergo different fluid contacting protocols. Thermal control is typically by slide pair, so that each slide pair undergoes the same temperature profile or different pairs can have different temperature programming. Small volumes of liquids can be manually applied to each of the slides. Each slide pair is provided with separate clamping mechanisms to seal DNA sample areas of each slide. Fluids are moved under negative pressure throughout the instrument, ensuring that no dangerous chemicals can be ejected under pressure. The present invention also provides for software control of fluid contacting and sample temperature using software running on an embedded personal computer. User input is by touchscreen, floppy disk drive. The system network distributes control signals and software instructions between master and satellite fluid control units and thermal control modules for each of the slide pairs.